Antigenic peptide fragments of vapa protein, and uses thereof

ABSTRACT

A chimeric GroEL protein is provided which includes a surface exposed to exogenous amino acid sequence, which comprises an antigenic determinant of, for example, a pathogenic micro-organism. The exogenous amino acid sequence might be inserted into a hydrophilic region of the GroEL protein to provide a means of exhibiting the antigenic determinant to elict an immune response specifically reactive to the antigenic determinant. This provides for a cellular bias to the elicited immune response and is thus likely to be particulary useful for intracellular parasites.

FIELD OF THE INVENTION

This invention relates to a GroEL chimeric protein and a vaccine whichcan be used in a method of eliciting an immune response in a mammal toan antigen, in particular to enhancing an immune response to amicroorganism or allergen by the providing a chimeric GroEL protein ornucleic acid with an inserted protein or nucleic acid of an immunogenicdeterminant for the microorganism or allergen.

BACKGROUND OF THE INVENTION

Certain microbial diseases of mammals are appropriately prevented byvaccinating a population at risk. The form of the vaccine hastraditionally been with killed, or attenuated live organism. Thisapproach has not been successful for all pathogens because antigenspresented at the time of infection may be masked or not present duringmass preparation of the vaccines. Additionally there are risksassociated with accidental exposure of the vaccinated population to liveorganism that were supposedly killed and adverse reactions to componentsof preparations made from whole organisms, in particular endotoxins.

To circumvent difficulties associated with the use of vaccines derivedfrom whole microorganisms, a major push over several decades has been toidentify specific antigenic determinants presented on the surface ofinfecting microorganisms, antibodies against which can reduce andpreferably eliminate the risk of infection. The aim is to identify oneor more major antigenic determinants which are stably present on themicroorganism for interaction with antibodies for opsonisation anddisposal by the immune system of the susceptible population; and toattempt to elicit an appropriate immune response to the one or moremajor antigenic determinant.

In this use of major antigenic determinants the hope is also to achievea response that is appropriate for the microorganism concerned. Thus animmune response biased toward cellular immunity is preferred for apathogen that is intracellular. Alternatively it might be referred toprovide for mucosal immunity should that present a particular barrier toentry by the pathogen.

Several important proteins have been identified as major antigens, orindeed have been identified as causal, such as toxins and frimbrialproteins in enteric pathogens. Whole proteins, or modified proteins havebeen used as the basis of vaccination trials. In some cases however theimmune response to immunisation programs have been rather low, andjudged as inadequate to provide a protective effect.

An understanding of specific antigenic determinants have been furtherrefined, and in particular in the case of some proteins to specificlinear amino acid sequences. This is the case for microorganisms as wellas for allergens. The hope is then to present these amino acid stringsin a format that elicits an immune response in the susceptiblepopulation. These linear antigenic determinants are, in one approach,inserted into another protein so as to be exposed at the surface andaccordingly result in a suitable immune response. There is uncertaintyin the nature of the immune response elicited by immunisation bypreparations containing such chimeric protein. One is not sure, forexample whether the type of response will be appropriate or if asufficiently strong immunity will be provided. Additionally an immunerespone to just one antigenic determinant may not be sufficient toprovide the protection required.

SUMMARY OF THE INVENTION

It has been found that an enhanced immune response was elicited bypresenting a chimeric protein into a target animal, the chimeric proteinis the result of the insertion of an antigenic determinant of thevirulence associated protein VapA into the GroEL2 protein of Rhodococcusequi.

This finding provides a promising approach to dealing with R. equiinfections in foals, however it also provides a mechanism of enhancingan immune response for other infections by utilising chimeric GroELproteins into which antigenic determinants, or haptenic antigens thatare normally poorly antigenic are inserted. The data, in particular toDNA vaccination, show an enhanced Th1 response, which is indicative of agreater cellular immunity compared to humoural immunity, and thus thegeneral approach may be particularly applicable to intracellularinfections. This finding also has implications beyond microbialinfections to eliciting immune responses more generally and can beapplicable, for example, to eliciting a hypoimmune response to variousantigens such as might be desired in the case of allergens.

Accordingly in a broad form of a first aspect the invention could besaid to reside in a method of eliciting an immune response in a mammalagainst an antigenic determinant, the method including the step ofproviding to the mammal a chimeric protein by a route and in a form toelicit an immune reaction that reacts with the antigenic determinant,the chimeric protein being a GroEL protein, modification or analoguethereof having a surface exposed exogenous amino acid sequence insertedtherein, said exogenous amino acid sequence reactive with antibodiesspecific to the antigenic determinant.

The invention is more particularly, however, applicable to microbialinfection and accordingly in a broad form of a second aspect theinvention could be said to reside in a method of eliciting an immuneresponse in a mammal against an antigenic determinant of amicroorganism, the method including the step of administering to themammal a chimeric protein, the chimeric protein being a GroEL protein,modification or analogue thereof having a surface exposed exogenousamino acid sequence inserted therein, said exogenous amino acid sequenceconfigured to elicit an immune response specifically reactive to theantigenic determinant of the microorganism.

In a broad form of a third aspect the invention could be said to residein a chimeric protein, said chimeric protein being a GroEL protein,modification or analogue thereof having a surface exposed exogenousamino acid sequence inserted therein, the exogenous amino acid sequenceconfigured to elicit an immune response specifically reactive to theantigenic determinant.

In a broad form of a fourth aspect the invention could be said to residein a nucleic acid encoding a chimeric protein, said chimeric proteinbeing a GroEL protein, modification or analogue thereof having a surfaceexposed exongenous amino acid sequence inserted therein, the exogenousamino acid sequence configured to elicit an immune response specificallyreactive to the antigenic determinant.

In a broad form of a fifth aspect the invention could be said to residein a compostion for eliciting an immune response in a mammal directedagainst a microorganism or allergen, the composition including achimeric protein in a pharmaceutically acceptable carrier, the chimericprotein being a GroEL protein, modification or analogue therof having asurface exposed exogenous amino acid sequence inserted therein, saidexogenous amino acid sequence reactive with antibodies specific to anantigenic determinant of the microorganism or allergen.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 Nucleotide and amino acid sequence [SEQ ID No 1] encoding GroEL2of Rhodococcus equi.

FIG. 2. Physical map of pIMVS-Re2. The R. equi groEL2 gene was insertedinto pET-28a (+) vector which expressed GroEL2 with 6 histidine residuesat the C-terminus. The vector contains a kanamycin cassette (Kan^(r))for the selection of transformants, T7 promoter and Lac operator (lacI)for induction of protein expression.

FIG. 3. Physical map of construct pcDNA3-Re1 (pcDNA3 containing R. equigroEL2 with modified Kozak sequence). Restriction sites used for cloninggroEL2 are indicated. The vector contains an ampicillin resistancecassette (Amp^(r)) for antibiotic selection, the human cytomegalovirusimmediate early promoter (Pcmv) and SV40 origin for episomalreplication.

FIG. 4 Physical map of construct pcDNA3-Re2 (Vector pcDNA3 with Kozaksequence containing R. equi vapA). Restriction sites used for cloningvapA are indicated. The vector contains an ampicillin resistancecassette (Amp^(r)) for antibiotic selection, the human cytomegalovirusimmediate early promoter (Pcmv) and SV40 origin for episomalreplication.

FIG. 5 Physical map of pIMVS-Re3. The R. equi vapA gene was insertedinto pET-28a (+) vector which expressed VapA with 6 histidine residuesat the C-terminus. The vector contained a kanamycin cassette (Kan^(r))for the selection of transformants, T7 promoter and Lac operator (lacl)for induction of protein expression.

FIG. 6 Schematic representation of overlap extension PCR performed tocreate the chimeric groEL2/vapA DNA vaccine construct pcDNA3-Re3. Theconstruct pcDNA3-Re1 was used as the template for the first two PCRreactions, in which products containing the inserted VapA epitope wereproduced (broken lines indicate sequence of VapA epitope). The PCRproducts obtained from these reactions were then used as templates forthe final reaction to produce the PCR product used to create vaccineconstruct pcDNA3-Re3.

FIG. 7 Amino acid sequence of R. equi GroEL2. Residues in bold indicateregion of hydrophilic residues associated with an area within GroELproteins significantly associated with immunogenicity (as described byother researchers) (Panchanathan, et al., 1998). The arrow indicates thepoint of insertion of the VapA immunogenic epitope NLQKDEPNGRA [SEQ IDNo 3] into GroEL2.

FIG. 8 Physical map of construct pcDNA3-Re3 (Vector pcDNA3 with Kozaksequence containing groEL2/vapA epitope chimeric gene). Restrictionsites used for cloning vapA are indicated. The vector contains anampicillin resistance cassette (Amp^(r)) for antibiotic selection, thehuman cytomegalovirus immediate early promoter (Pcmv) and SV40 originfor episomal replication.

FIG. 9 Physical map of pIMVS-Re4. The chimeric groEL2/vapA gene wasinserted into pET-28a (+) vector which expressed the protein with 6histidine residues at the C-terminus. The vector contained a kanamycincassette (Kan^(r)) for the selection of transformants, T7 promoter andLac operator (lacI) for induction of protein expression.

FIG. 10 R. equi GroEL2 specific IgG1/IgG2a antibody response followingimmunization with pcDNA3 vector (control), pcDNA3-Re1, His-tagged GroEL2protein, pcDNA3-Re1+pORF-mlLI2 or live R. equi vaccine. Antibody levelswere determined 2, 4 and 6 weeks after initial immunization. Data areshown as mean and standard error. Data were analysed using the Wilcoxon(rank sum) two-sample test (P<0.05) (FIG. 10A) IgG1 response: allvaccine constructs elicited a statistically significant responsecompared to the control (FIG. 10B) IgG2a response: all vaccineconstructs elicited a statistically significant response compared to thecontrol. pcDNA3 vector (control) (−), pcDNA3-Re1 ♦, His-tagged GroEL2protein ▴, pcDNA3-Re1+pORF-mIL12 ▪, live R. equi ATCC 33701 ★

FIG. 11 R. equi GroEL2 specific IgG2b antibody response and DTH responsefollowing immunization with pcDNA3 vector (control), pcDNA3-Re1,His-tagged GroEL2 protein, pcDNA3-Re1+pORF-mIL12 or live R. equivaccine. IgG2b subclass antibody levels were determined 2, 4 and 6 weeksafter initial immunization. DTH response was determined two weeks afterthe last boost. Data are shown as mean and standard error. Data wereanalysed using the Wilcoxon (rank sum) two sample test (P<0.05) (C)IgG2b response: all vaccine constructs elicited a statisticallysignificant response compared to the control (D) Delayed typehypersensitivity (DTH) response: all vaccine constructs elicited astatistically significant response compared to the control

pcDNA3 vector (control) (−), pcDNA3-Re1 ♦, His-tagged GroEL2 protein ▴,pcDNA3-Re1+pORF-mIL12 ▪, live R. equi A TCC 33701 ★

FIG. 12 R. equi VapA specific IgG1/IgG2a antibody response followingimmunization with pcDNA3 vector (control), pcDNA3-Re2, His-tagged VapAprotein, pcDNA3-Re2+pORF-mlLI2 or live R. equi vaccine. Antibody levelswere determined 2, 4 and 6 weeks after initial immunization. Data areshown as mean and standard error. Data were analysed using the Wilcoxon(rank sum) two sample test (P<0.05) (FIG. 11A) IgG1 response: allvaccine constructs elicited a statistically significant responsecompared to the control (FIG. 11B) IgG2a response: all vaccineconstructs elicited a statistically significant response compared to thecontrol. pcDNA3 vector (control) (−), pcDNA3-Re2 ⋄, His-tagged Vap Aprotein ♦, pcDNA3-Re2+pORF-mIL12 ▪, live R. equi ATCC 33701 ★

FIG. 13 R. equi VapA specific IgG2b antibody response and DTH responsefollowing immunization with pcDNA3 vector (control), pcDNA3-Re2,His-tagged VapA protein, pcDNA3-Re2+pORF-mlLI2 or live R. equi vaccine.IgG2b antibody levels were determined 2, 4 and 6 weeks after initialimmunization. DTH response was determined two weeks after the lastboost. Data are shown as mean and standard error. Data were analysedusing the Wilcoxon (rank sum) two sample test (P<0.05) (FIG. 12A) IgG2bresponse: all vaccine constructs elicited a statistically significantresponse compared to the control (FIG. 12B) Delayed typehypersensitivity (DTH) response: all vaccine constructs elicited astatistically significant response compared to the control. pcDNA3vector (control) (−), pcDNA3-Re2 ⋄, His-tagged VapA protein ♦,pcDNA3-Re2+pORF-mIL12 ▪, live R. equi ATCC 33701 ★

FIG. 14 R. equi GroEL2 specific IgG1/IgG2a antibody response followingimmunization with pcDNA3 vector (control), pcDNA3-Re3, His-taggedchimeric GroEL2/VapA protein, pcDNA3-Re3+pORF-mIL12 or live R. equivaccine. Antibody levels were determined 2, 4 and 6 weeks after initialimmunization. Data are shown as mean and standard error. Data wereanalysed using the Wilcoxon (rank sum) two sample test (P<0.05) (FIG.14A) IgG1 response: all vaccine constructs elicited a statisticallysignificant response compared to the control (FIG. 14B) IgG2a response:all vaccine constructs elicited a statistically significant responsecompared to the control. pcDNA3 vector (control) (−), pcDNA3-Re3 ⋄,His-tagged chimeric GroEL2/VapA protein ♦, pcDNA3-Re3+pORF-mlLI2 ▪, liveR. equi ATCC 33701 ★

FIG. 15 R. equi GroEL2 specific IgG2b antibody response and DTH responsefollowing immunization with pcDNA3 vector (control), pcDNA3-Re3,His-tagged GroEL2/VapA protein, pcDNA3-Re3+pORF-mlLI2 or live R. equivaccine. IgG2b subclass antibody levels were determined 2, 4 and 6 weeksafter initial immunization. DTH response was determined two weeks afterthe last boost. Data are shown as mean and standard error. Data wereanalysed using the Wilcoxon (rank sum) two sample test (P<0.05) (FIG.15A) IgG2b response: only the chimeric protein vaccine constructelicited a statistically significant response compared to the control(FIG. 15B) Delayed type hypersensitivity (DTH) response: all vaccineconstructs elicited a statistically significant response compared to thecontrol, pcDNA3 vector (control) (−), pcDNA3-Re3 ♦, His-tagged chimericGroEL2/VapA protein ▴, pcDNA3-Re3+pORF-mIL12 ▪, live R. equi ATCC 33701★

DETAILED DESCRIPTION OF THE INVENTION

By way of a shorthand notation the following three and one letterabbreviations for amino acid residues are used in the specification asdefined in Table 1.

Where a specific amino acid residue is referred to by its position inthe polypeptide of an protein, the amino acid abbreviation is used withthe residue number given in superscript (i.e. Xaa^(n)) TABLE 1Three-letter One letter Amino Acid Abbreviation Abbreviation Alanine AlaA Arginine Arg R Asparagine Asn N Aspartic Acid Asp D Cysteine Cys CGlutamine Gln Q Glutamic acid Glu E Glycine Gly G Histidine His HIsoleucine Ile I Leucine Leu L Lysine Lys K Methionine Met MPhenylalanine Phe F Proline Pro P Serine Ser S Threonine Thr TTryptophan Trp W Tyrosine Tyr Y Valine Val V

GroEL proteins were originally identified in E. coli as one of the hostfactors required for bacteriophage capsid assembly during a lyticinfection. The groEL2 gene is highly conserved between species and hasbeen used in phylogenetic research (Gupta, 1995, Gupta, 2000). Membersof the GroEL family are found in all eubacterial cells as well aseukaryotic mitochondria and chloroplasts (Gupta, 1995).

GroEL is known to facilitate the correct folding of various bacterialproteins as well as prevent the aggregation of denatured proteins by anATP dependent mechanism (Craig, et al., 1993). The GroEL protein iscomposed of 14 subunits, arranged in heptameric rings with a centralcavity. This central cavity referred to as the ‘Anfinsen cage’ providesa shielded environment for the refolding of proteins (Ma, et al., 2000).

In most eubacteria, the groEL (L-large) gene that encodes a proteinapproximately 60-65 kDa in size is present in the groE operon togetherwith a smaller protein (Hsp10) encoding groES (S-small) gene (Segal andRon, 1996). Several organisms contain just one copy of the groEL gene inan operon (Segal and Ron, 1996). However, there are several organismsincluding Mycobacteria sp. (Rinke de Wit, et al., 1992) and notablyα-proteobacteria, such as the nitrogen fixing soybean nodule bacterium,Bradyrhizobium japonicum, that possess two or more copies of GroELencoding genes in their chromosome (Karlin and Brocchieri, 2000).Organisms such as Mycobacteria sp. and other actinomycetes contain twogroEL genes. One of these is designated groEL2 and is usually notassociated with a groES gene in a bicistronic operon. Similar to groEL1which is associated with groES in an operon, groEL2 is also inducedfollowing heat shock and other physiological stress (Duchêne, et al.,1994, Mazodier, et al., 1991).

The expression of GroEL proteins are known to be upregulated duringinfection of a host in a range of bacterial pathogens (Noll, et al.,1999). Importantly, these proteins have been shown to be immunodominantantigens in both humoral and cell-mediated host responses against manybacteria, particularly with respect to intracellular pathogens such asLegionella pneumophila (Sampson, et al., 1986, Zügel and Kaufmann,1999b).

GroEL2 appears to be transcribed at a much higher rate than groEL1 whenthe organism is subjected to environmental stress such as hightemperature (de León, et al., 1997). In addition, the groEL2 encodedprotein appears to be immunodominant over the groEL1 encoded protein,although antibodies to both proteins are observed during infections(Lathigra, et al., 1991. Rinke de Wit, et al., 1992, Shinnick, 1991).

GroEL has been used on its own as a means of immunising againstMycobacterium tuberculosis as a preventative for tuberculosis in humans(Lowrie et al., 1997, 1999)

GroEL has been used in conjugates to enhance the immune response inpoorly immunogenic antigens Cohen et al U.S. Pat. No. 5,869,058.

The present invention utilises the immunogenic properties of GroEL bythe insertion of an exogenous amino acid sequence that is reactive withantibodies to an antigenic determinant of a surface protein of themicroorganism into one or more regions of the GroEL protein that lead tois surface exposure on the chimeric protein. The inventors are unawareof any other demonstration of an enhanced immune reaction using thisapproach.

This approach has a range of applications. In one specific aspect theapplication is limited to infection of foals by Rhodococcus equii, butin other broader aspects the invention is to a range of pathogens, othermicroorganism or indeed other antigenic determinants such as might bepresent in a range of allergens.

It is preferred that the GroEL into which the exogenous amino acidsequence carries at least a major antigenic determinant that isimmunogenically identical to the GroEL carried by the species ofmicro-organism with respect of which an immune response is to beelicited. Not all of the major antigenic determinants of the speciesspecific GroEL need be carried. The preference is therefore that theimmune response elicited in the mammal will be directed in part also toGroEL antigenic determinants. The most efficient way of providing such aGroEL is to make the insertion of the exogenous amino acids into theGroEL of the microorganism for which immunity is desired, to form thechimeric protein. Thus for example where it is desired to induce animmune reaction in R. equi, then the GroE1 from R. equi is used inaddition to an amino acid sequence derived from R. equi.

Whilst it is desirable that the specificity of the immune response isdirected totally to major antigenic determinants, given the conservativenature of the GroEL it is likely that the species specificity of theGroEL will not particularly influence the manner in which the antigenicdeterminant within the exogenous amino acid sequence is presented in themammal.

The GroEL forming the basis of the chimeric protein might be any oneencoded by a pathogenic micro-organism. There microorganism might beselected from the group of micro-organisms comprising Listeria ivanovii,Listeria monocytogenes, Salmonella enterica, Bordatella species,Mycobacterium species, Nocardia Species, Shigella species,Enteropathogenic E. coli, Yersinia species, Legionella species,Francisella tularensis, Brucella species, Chlamydiae, Rickettsiae.

DNA sequences of many GroEL genes are known many are referred to inGupta, 1995 and Gupta 2000, and in Richardon et al., (1998) and Saibil(2000). Specific examples of DNA/amino acid sequences are listed asfollow Mycobacterium marinum (genbank U55831), Mycobacteriumtuberculosis H37Rv (genbank AL021932), Mycobacterium bovis (genbankM17705), Mycobacterium avium (genbank AF281650), Tsukamurellatyrosinosolvens (genbank U90204) Rhodococcus equi (genbank AF233387),Streptomyces lividans (genbank X95971), Streptomyces albus (genbankM76658), Corynebacterium aquaticum (genbank AF 184092), Pseudomonasaeruginosa (genbank M63957), Helicobacter pylori (genbank X73840),Borrelia burgdorferi (genbank X65139).

Where organisms carry two groEL genes preferably the groEL gene is thatgene expressing groEL2. Such organism might include Mycobacteria speciesand other actinomycetes and α-proteobacteria.

It is also anticipated that variations and modification of the GroELproteins will also form a reasonable basis for chimeric proteins. It isknown that conservative substitutions in proteins, in particular ininessential parts of the protein still permit function, and indeed themammal into which these chimeric proteins are to be introduced do notdepend on them for GroEL function, and therefore the substitutions atleast in certain parts need not be structural. It is anticipatedtherefore that groEL genes that have had genetic modification such aspoint mutations, and rearrangements such as deletions, truncations,substitutions, inversion and duplication may still function toappropriately present the inserted exogenous amino acid sequence andpreferably also one or more major GroEL antigenic determinant. Forexample the GroEL protein might include partial deletion of an existinghydrophilic region or amino acid string defining an antigenicdeterminant to so that the exogenous amino acid sequence can bepartially or fully substituted therein. In this respect it is preferablythat the chimeric protein is still able to form the double heptamericring, to thereby provide for a substantial exposure of the exogenousamino acid sequence.

The exogenous amino acid sequence as indicated above is to be insertedin the GroEL amino acid sequence so as to be exposed to the surface ofthe chimeric protein. Thus the exogenous amino acid sequence ispresented so as to be accessible to receptors responsible for inducingthe desired immune response. More preferably as indicated above theexogenous amino acid sequence is exposed to the surface of the doubleheptameric ring structure into which GroEL is formed.

One approach to determining appropriate sites for insertion is tocalculate from the predicted amino acid sequence of the proteinconcerned a hydrophobicity plot, and to insert the exogenous amino acidsequence into one or more of the hydrophilic regions. Thus it might bedesired to select more than one site of insertion. Indeed where themicroorganism concerned has more than one major antigenic determinant,it might thus be desired to form a multivalent insertion, to express twoor more exogenous amino acid sequences providing two or more furtherantigenic determinants.

Another approach to this is to insert the exogenous amino acid sequencesinto the GroEL sequence known itself to be an antigenic determinant.These may be as identified in Panchanathan et al., (1998). This latterapproach however may not necessarily be preferred because it may be morepreferable to have both the major existing antigenic GroEL determinantsas well as that provided by the exogenous amino acid sequence so thattwo antigenic determinants are presented for an immune response.

With respect of the Rhodococcus equi GroEL, the hydrophilic regionsmight be selected from one or more of the following: —V26-S54, V73-T90,G109-A144, M191-L246, R270-1290, G342-A397 and V415-N468

More particularly this might be M191-L246.

These or similar hydrophilic regions in other GroEL protein can bepredicted using the methods of Hopp and Woods. (1981) which method canalso be used to predict which amino acids might be an antigenicdeterminant.

Assistance in that regard can also be had to the extensive work that hasbeen made in amino acid/nucleic acid comparison and model work for GroEL(Richardon et al., 1998; Saibil, 2000). Using these models and/orprotein sequence comparisons one can make a relatively informedcalculation as to one or more sites most likely to provide for goodpresentation of the exogenous amino acid sequence. Additionally theabove two references can assist in selecting an appropriate GroELsequence to use for with a particular micro-organism or other targetantigen.

The insertion might be in the form of a direct insertion into theexisting sequence. Thus should the exogenous amino acid sequence be 11amino acids long the chimeric protein will be 11 amino acids longer thanthe GroEL on which the chimeric protein is based. Alternatively deletionof GroEL of some amino acids might be effected in addition to theinsertion of the exogenous amino acid sequence, to keep the size of anysurface exposed loop down or perhaps the same size as they originallywere.

Selection of the exogenous amino acids is anticipated to be quiteimportant. Typically it is anticipated that they would represent linearantigenic determinants. Antigenic determinants on the surface of aprotein are those features that are capable of binding an antibody. Attimes there is sufficient binding to a sequence of amino acids in alinear string, such that the string of amino acids presented will elicitbinding by an antibody. These antigenic determinants are known as linearantigenic determinants. Alternatively more than one string ofconsecutive amino acid sequences are required to bind an antibody, thesemore complex antigenic determinants arise where antibodies recognisedamino acids adjacent to one another on the surface of a protein but byreason of the folding of the primary amino acid sequence are notadjacent on the same linear sequence. The present invention is mostparticularly concerned with the presentation of the more simple linearantigenic determinants. Although where perhaps two strings of aminoacids sequences form an antigenic determinants and the spacing of theadjacent loops in the GroEL matches that of the original antigenicdeterminant the present invention may be adapted for that purpose.

The length of linear antigenic determinant varies considerably and mayrange from three or four amino acid to about 25 amino acids.

The amino acid sequence might be selected to be reactive with a majorantigenic determinant of a pathogenic micro-organism. It is found thatwith infections of a particular strain of micro-organisms that commonlyan immune response is directed to just a few antigenic determinants. Oneantigenic determinant may in fact dominate. Additionally there mightsome minor antigenic determinant that are recognised. Generally themajor antigenic determinant are those that are more accessible to cellsof the immune system including those responsible for recognising anddisposing of infectious micro-organisms. It is desirable to induceimmunity as against major antigenic determinant because it isanticipated that these will provide, in a vaccine, for a betterprotective effect, or in the case of an allergen provide for moreeffective tolerance. Indeed in the case of inducing tolerance to anallergen it is anticipated that the antigenic determinant used will bethe one to which the individual concerned has an allergic reaction.

The exogenous amino acids sequence might therefore be selected from alarge range of currently identified major antigenic determinants. Theymight be selected for example from the following micro-organisms.Listeria ivanovii, Listeria monocytogenes, Salmonella enterica,Bordatella species, Mycobacterium species, Nocardia Species, Shigellaspecies, Enteropathogenic E. coli, Yersinia species, Legionella species,Francisella tularensis, Brucella species, Chlamydiae, Rickettsiae.

Other examples of suitable exogenous amino acids sequences contemplatedby the present invention are as follows. They may be derived fromviruses such as rhinoviruses, rotavirus, retroviruses, polivirus.Suitable antigenic determinants for HIV might be those identified byEnshell-Seijffers et al., (FASEB 2001 15; 2012-2020). Hepatitis C virussuch as identified for the E2 glycoprotein by Bugli et al., (J. Virol.2001 75:9986-9990). Hepatitis delta virus identified by Fiedler andRoggendorf (Intervirology (2001) 44:154-161).

Similarly these might be for vaccination against various allergens forexample certain pollen antigens identified by Focke et al., (FASEB(2001) 15:2042-2044).

Other amino acid sequences suitable for this invention might includethose referred to in the review of random peptide libraries by Irving,Pan and Scott (Current Opinions in Chem Biol (2001) 5:314-324), or inthe review by Partido (2000, Current Opinions in Molecular Therapy2:74-79).

Indeed the exogenous amino acid sequence might additionally be desiredto be a GroEL sequence. Thus, for example it might be desired toprovided for an enhanced immune reaction by providing for more than onecopy of a GroEL specific antigenic determinant.

Rhodococcus equi is an encapsulated and rod shaped, Gram positivebacterium that is considered to be a soil saprophyte that survives wellin the soil environment. R. equi has long been considered a pathogen inhorses principally in foals fewer than 6 months old (particularly 1-3months old). Infection by the organism is accompanied by extra-pulmonarymanifestations, causes a pyogranulomatous pneumonia, often such asbacteraemia, lymphadenitis, meningitis and enteritis (Barton and Hughes,1980; Giguere and Prescott, 1997; Takai, 1997). Infections are oftenfatal if untreated. Apart from causing disease in horses, R. equi alsocauses infections in cattle, pigs and goats (Barton, 1992). R. equi isalso known to cause severe pulmonary and disseminated disease inimmuno-compromised humans, particularly AIDS patients (Capdevila et al.,1997).

In Australia most equine R. equi infections occur in summer (December toFebruary) when the age of the foals as well as the warm and dryenvironmental conditions make the animals more susceptible to infection(Barton and Hughes, 1984).

Many vaccine candidates have been tested for the prevention of R. equiinfection in foals. Vaccine candidates have predominantly been proteinsubunit or whole cell preparations. A vaccine preparation known as‘Rhodovac’ developed by Clínica Equina (Capitán Sarmiento, Argentina),contains high concentrations of soluble virulent R. equi antigensincluding VapA (Becú, et al., 1997). Other VapA containing antigenpreparations have also been developed (Prescott, et al., 1997a). Inaddition, a range of vaccine preparations comprising killed or live R.equi (Prescott, et al., 1997b, Varga, et al., 1997) have also beentested.

R. equi produces a range of putative virulence factors such ascholesterol oxidase, phospholipase C and Iccithinase (Smola et al.1994). However one of the more important putative virulence factors isconsidered to be a 17 kDa virulence associated protein (VapA) which isplasmid encoded. This protein is known to be produced by up to 90% ofequine clinical isolates of R. equi. Although VapA producing strains arewidespread among disease causing isolates, recent work has shown thatVapA protein alone is not sufficient to cause disease in foals and thatother as yet unknown plasmid borne factors are likely to be involved(Giguere et al., 1999). The role of VapA in virulence is yet to beelucidated, although there is strong evidence to suggest that theplasmid encoding the protein may play an important part in the survivalof the organism within macrophages (Hondalus and Mosser, 1994).

In one specific form the exogenous amino acids might be the antigenicdeterminant found to be dominant with respect of Rhodococcus equi beinga part of the VapA protein (Vanniasinkam et al., 2001).

A putative 20 amino acid region of the VapA protein that is recognisedby antibodies in the sera of horses infected with R. equi has beenidentified as TSLNLQKDEPNGRASDTAGQ [SEQ ID No 2], although it will beunderstood that the minimal region for antigenic recognition may befurther defined within the identified sequence, or additionally theidentified sequence may contain two or more separate adjacent epitopes.Thus the amino acid sequence may be any peptide that is capable ofmimicking this region in so far as providing VapA specificimmunogenicity. Therefore the peptide may be part of a larger peptidethat contains the amino acid sequence TSLNLQKDEPNGRASDTAGQ [SEQ ID No 2]of the present invention, as well as one or more of the amino acidseither side of that sequence in the native VapA protein.

Therefore in one form of the this aspect the amino acid sequence has 5or more amino acid residues and contains all or part of the sequenceTSLNLQKDEPNGRASDTAGQ [SEQ ID No 2], or immunologically active derivativeor analogue thereof. Preferably the peptide contains 7 to 30 amino acidresidues, and more preferably 10 to 12 amino acid residues. Mostpreferably the peptide contains the sequence NLQKDEPNGRA [SEQ ID No 3].

Whether a peptide of the present invention provides for VapA specificimmunogenicity can be determined routinely by following the proceduresset out in (Vanniasinkam et al 2001).

The peptide in this aspect of the invention may also be homologous toany of the abovementioned peptides provided that the peptide providesfor VapA specific immunogenicity. In this context, a peptide isconsidered homologous to a peptide of the present invention when it isimmuno cross-reactive with antibodies specific for the R. equi VapAprotein. It will be recognised by those skilled in the art that someamino acid sequences within the peptide can be varied withoutsignificant effect on the structure or function of the peptide. Thus forinstance it is anticipated that ‘type’ amino acid substitutions stillretain immuno cross reactivity and as such a neutral amino acid may beconservatively substituted with another neutral natural or non-naturalamino acid, an acidic amino acid may be conservatively substituted witha natural or non-natural acidic amino acid, a hydrophilic amino acid maybe substituted with another hydrophilic amino acid, and so on, providedthat the immunological function of the peptide is not altered by thesubstitution.

Typically seen as conservative substitutions are the replacement of onefor another among the aliphatic amino acids Ala, Val, Leu and Ile;interchange of the hydroxyl residues Ser and Thr; exchange of the acidicresidues Asp and Glu; substitutions between the amide residues Asn andGin; exchange of the basic residues Lys and Arg; and replacements amongthe aromatic residues Phe and Tyr. Preferably the homologous peptideshares 50% homology with a peptide of the present invention, morepreferably shares 70% homology, and most preferably shares 90% homology.

Generally the insertion will be achieved at the nucleic acid level.Using purified DNA of a vector encoding the GroEL protein, synthesizinga DNA sequence encoding the exogenous amino acid sequence, cutting theDNA encoding the GroEL protein at the site of insertion using arestriction endonuclease, ligating in the synthetic sequence, andisolating the recombinant DNA molecule so formed and introducing it intoan appropriate host or vector to amplify the DNA for a DNA vaccine orfor the production of a recombinant protein preparation.

It is found that the cellular immunity (Th1 immunity) is enhanced by theuse of this invention. Accordingly it is anticipated that this may be aparticularly useful approach to vaccinating for intracellular pathogens.To further-bias the immune reaction in favour of Th1 immunity it mightbe desired to provide the vaccine as a nucleic acid vaccine.

There are various strategies, which may improve the protective efficacyof the vaccines of this invention, and many of them are well knownincluding the use of various adjuvants. Research has shown that theco-administration of immunostimulatory molecules, such as TL-18 (Kim, etal., 2001), together with a pathogen specific antigen encoding DNAvaccine may be used to enhance a Th1 type protective response. Thisapproach may be useful to improve the level of Th1 type immunityobserved with the vaccines used in this study, in order to induce asufficient immune response that would confer protection against R. equi.Other strategies to improve the protective efficacy of the vaccine wouldinclude the development of multi-epitope vaccines that contain other R.equi genes in addition to groEL2 or the use of a prime-boost strategy toimmunise the host (Ramshaw and Ramsay, 2000). Il-12 is also recognisedfor its role in maintaining long term cell-mediated immunity (Park andScott, 2001). Therefore coadministration of IL-12, as a DNA vaccine orrecombinant protein, in conjunction with a suitable primary vaccine mayenhance protection against R. equi in the host.

The chimeric protein might be admininistered following purification ofthe protein and forming a vaccine composition that is administered tothe mammal to elicit an immune reaction. The purification might be byknown methods. The chimeric protein will be encoded by any one of anumber of known expression vectors that is introduced into an expressionmicroorganism. Purification by known methods follows fermentation. Thepurified or semi purified protein can then be administered.

The administration might be parenteral such as subcutaneously,intramuscularly, or it might simply presented to a mucosal surface,perhaps by pulmonary administration or alternatively administrationmight be intraperitonealy. The mucosal administration may be aimed atinducing local mucosal immunity to provide a barrier to entry by theorganism concerned.

Preferably the chimeric protein is administered in pharmaceutical dosageform as a composition or formulation comprising an immunogenicallyeffective amount of the chimeric protein. The amount of chimeric proteinadministered will vary depending on the pharmacokinetic parameters,severity of the disease treated or immunogenic response desired Dosesmay be set by a physician or veterinarian considering relevant factorsincluding the age, weight and condition of the vertebrate including, inthe case of immunogenic dosage forms, whether the vertebrate has beenpreviously exposed to the microorganism responsible for the disease tobe vaccinated against as well as the release characteristics of thepeptide from pharmaceutical dosage forms of the present invention.

The composition may be injected or may be added to a pharmaceuticallyacceptable carrier as will be apparent to those skilled in the art andas set out in “Remington's Pharmaceutical Sciences”, Sixteenth Edition,Mack Publishing Co, 1980, and include water and other polar substances,including lower molecular weight alkanes, polyalkanols such as ethyleneglycol, polyethylene glycol and propylene glycol as well as non-polarcarriers.

The method of administering the vaccine may vary and could includeintravenous, buccal, oral, transdermal and nasal as well asintramuscular or subcutaneous administration.

Preferably, the vaccine is administered by inhalation which may then setup local immunity. Alternatively the vaccine may be administered usingother forms of mucosal priming.

In particular with the GroEL/vapA chimeric protein it might be desiredto administer to the pulmonary system, perhaps as an aerosol, becausethat represents the transmission route of the pathogenic organism.

The vaccine might be provided in a composition using the chimericprotein, however, in another form the chimeric protein can be providedto the mammal by the injection of a nucleic acid encoding the chimericprotein preferably carried in a suitable nucleic acid vector. In thatform the nucleic acid, usually DNA vector, is typically introducedintramuscularly, by published methods. Some of the nucleic acid isintroduced intracellularly to transform a cell. The transformed cellthen expresses the chimeric protein which is presented either on thecell surface to induce the immune reaction or is presented on senescenceof the transformed cell to elicit an immune reaction.

DNA vaccines have been used for the induction of long-term cellularimmunity for the prevention of bacterial and viral infections in largeanimals such as cattle (Babiuk, et al., 1998, Chaplin. et al., 1999).Lowrie et al., 1997. 1999 have used this approach to immunise againsttuberculosis utilising, a GroEL based DNA vaccine. This might alsorequire assistance with a substance such as Bupivacaine to assist withuptake of the DNA.

Alternatives for enhancing DNA vaccine delivery include methods ofvaccine delivery such as gene gun inoculation (Yoshida, et al., 2000),the use of attenuated bacteria as the vaccine carrier (Dietrich, et al.,2001) or electrotransfer of the plasmid (Bachy, et al., 2001). Inaddition, the co-administration of adjuvants such as Th1response-promoting cytokines or cationic mannan-coated liposomes (Toda,et al., 1997) could also be tested. Alternatively, the plasmid DNA couldbe administered as a supercoiled molecule (minicircle) devoid of originof replication and antibiotic resistance cassettes. Minicircles aresmaller and potentially safer than many currently used vaccine vectorsand importantly have been shown to exhibit a high level of expression invivo (Darquet, et al., 1999).

Potential DNA vaccines against various bacterial pathogens, particularlyintracellular pathogens such as Mycobacterium tuberculosis (Lowrie, etal., 1997) and Chlamydia psittaci (Vanrompay, et al., 1999) have beendeveloped. It is postulated that since intracellular pathogens ingenerally require a Th1 type response for protective immunity, a DNAvaccine approach, which may be used to elicit a Th1 response, may bepotentially more useful than subunit vaccines (Strugnell, et al., 1997)or attenuated live vaccines which can sometimes be subject tovariability in efficacy as has been observed with Mycobacterium bovisbased vaccines (Behr and Small, 1997). Often, genes used in DNA vaccinesare selected following the identification of immunodominant antigens andthe genes encoding them. Some of the proteins encoded by these vaccinecandidates genes have included heat shock proteins (Lowrie, et al.,1997) (Svanholm, et al., 2000), secreted antigens (Kamath, et al., 1999)and a range of other immunogens such as outer membrane proteins (Pal, etal., 1999).

The present invention is further illustrated by the followingexemplification which is not intended to be limitng in any way.

EXAMPLES

Methods

General molecular biological methods were employed to undertake themicrobiological techniques and molecular miological techniques are asgenerally set out in Sambrook et al., 1989.

Example 1 Cloning and Sequencing of R. equi of Groel

Strains and Plasmids

R. equi ATCC 6939 was used for the cloning and sequencing of groEL2. Thevector pET-28a(+) (Novagen) was used for the production of histidine(His)-tagged GroEL2 in E. coli BL21 (DE3).

Bacterial Growth Conditions

Bacteria were grown in L-broth for protein expression. Columbia agar wasused for the propagation of transformants. E. coli DH5α was used as ahost for recombinant plasmids for the cloning of groEL2 and E. coli BL21(DE3) was used for the expression of His-tagged GroEL.

PCR amplification of a groEL2 gene containing fragment of the R. equichromosome Oligonucleotide primers were designed to amplify a fragmentof the R. equi groEL2 gene. Sequences of these primers were based uponregions of homology between the published sequences of the groEL2 ofMycobacterium tuberculosis H37 Rv (GenBank Accession No: AL021932) andgroEL of Tsukamurella tyrosinosolvens (GenBank Accession No: U90204).(these genes were chosen based upon 16S r-RNA studies indicating thatTsukamurella and Mycobacterium sp. are closely related to R. equi (Ruimyet al., 1995) and therefore likely to possess a groEL2 gene highlysimilar to the R. equi groEL2 gene).

The forward primer used was 5′-CAAGGAGGTCGAGACCAAGG-3′ [SEQ ID No 4] andreverse primer was 5′-GTGCCGCGGATCTTGTTGAC-3′ [SEQ ID No 5]. PCRamplification was carried out, using an annealing temperature of 64° C.and chromosomal DNA from R. equi as the template. The PCR product wassequenced and Digoxigenin labelled (Boehringer Mannheim, Germany). Thelabelled product was then used to probeR. equi chromosomal DNA digestedseparately with the following restriction enzymes in Southern blotanalysis: SacI, XbaI, SmaI, EcoRI, BamHI, NsiI, HindIII, KpnI and SphI.Fragments of differing lengths were identified in R. equi chromosomalDNA depending upon the digesting enzyme. A single SphI fragment 4.7 kbin size was identified and cloned into pGEM-7Zf(−) (construct wasdesignated pIMVS-Re1) and the nucleotide sequence of the R. equi insertdetermined.

Sequencing of pIMVS-Re1 was performed using ABI Prism Big Dye chemistry(PE Applied Biosystems).

Expression of GroEL2 in R. equi

Two 10 ml aliquots of R. equi were grown overnight at 30° C. in L-brothwith shaking. The following morning each aliquot culture was incubatedat 30° C. or 42° C. for two hours with shaking. The culture was thencentrifuged at 17, 000 g for 15 mins and pellet was resuspended in 1 ml1×PBS and sonicated for 1 min and prepared for western immunoblotanalysis using the Chlamydia trachomatis Hsp60 monoclonal antibody(Affinity Bioreagents Inc., CO, USA). This antibody is specific foramino acids 517 to 522 of the C. trachomatis HSP60 amino acid sequenceand is known not to cross react with E. coli HSP60. The first fiveresidues of the immunoreactive epitope (LTTEAL) [SEQ ID No 6] of themonoclonal antibody were identical to the amino acid residues 512 to 516of the R. equi GroEL2 sequence (FIG. 1), and was therefore predicted todetect this protein when used in a western immunoblot analysis.

Sequence analysis was performed using GeneBase version 1.0 (AppliedMaths, Kortrijk, Belgium) and BLASTX version 2.0 (Altschul, et al.,1997). The promoter region of the groEL2 gene was analysed using, theBerkeley Drosophila Genome Project promoter prediction website(www.fruitfly.org).

Subcloning of the R. equi groEL2 Gene

The groEL2 gene was PCR amplified using a forward primer5′-ACGGTACCATGGCCAAGATCATCGC-3′[SEQ ID No 7] containing an introducedNcoI site (underlined) and reverse primer 5′-CGTC{overscore(AAGCTT)}GAAGTCCATGCCGC-3′[SEQ ID No 8] containing an introduced HindIIIsite (underlined) for cloning into pET-28a (+) (Novagen) expressionvector. PCR was performed under standard conditions using DyNAzyme™ EXTDNA polymerase, an annealing temperature of 61° C. using a CsCl gradientpurified preparation of pIMVS-Re1 as template. The PCR product andvector were separately digested with NcoI and HindIII and ligated tocreate construct pIMVS-Re2 (FIG. 2).

Production of a C-terminal His-Tagged GroEL2 Protein

Plasmid pIMVS-Re2 was transformed into E. coli BL21 (DE3) and theHis-tagged GroEL2 protein was expressed and purified using Ni²⁺-NTAagarose (Qiagen) by the following method.

The clone containing pIMVS-Re2 was grown overnight in 4 ml L-brothcontaining 50 μg/ml kanamycin. This culture was added to 200 ml ofL-broth containing 50 μg/ml kanamycin and was incubated with shaking for3 hours. Protein production was induced with the addition of IPTG to thefinal concentration of 1 mM and the culture was grown for another 4hours under the same conditions. The culture was then centrifuged at3000 g and the pellet was stored at −20° C. overnight. The followingday, the pellet was resuspended in 10 ml lysis buffer (20 mM Tris, pH8and 1001 nM NaCl) and sonicated using 5, 15 second pulses. The solutionwas centrifuged at 17, 000 g for 15 mins. The supernatant was added to 1ml Ni-NTA agarose that had been washed twice in lysis buffer (10 mlbuffer added to the Ni-NTA agarose and centrifuged at 17, 000 g for 1min). The solution was mixed on a rotary mixer (200 rpm) for 2 hours.The solution was then loaded on to a 5 ml column (Qiagen) and the columnflow-through was removed. The Ni-NTA slurry deposited in the column waswashed twice with 5 ml lysis buffer (buffer was added to the column andallowed to empty by gravity). A 500 μl aliquot of 250 mM imidazolesolution was added to elute the protein from the NI-NTA slurry(performed twice), the protein eluate was stored in 100 μl aliquots at−20° C. until required.

Whole cell and protein preparations were separated on a 10% SDS PAGE.

N-terminal Sequence Analysis of C-Terminal 6× His-Tagged GroEL2

The 100 μl of purified His-tagged GroEL2 protein was separated on 10%SDS-PAGE and transferred to polyvinylidene difluoride membrane(Immobilon-P, Millipore, Mass., USA). The protein was then subjected toN-terminal amino acid sequencing by the Edman Degradation method (thesequencing was carried out by the Australian Proteome Analysis Facility,Macquarie University, NSW, Australia).

Results

The oligonucleotide primers amplified a 402 base pair (bp) PCR productfrom R. equi. This PCR product was found to be partially homologous tothe groEL2 sequence of Mycobacterium avium and Mycobacteriumparatuberculosis (P=6×10⁻⁶¹) and was used as a probe in Southernhybridisation to identify a transformant possessing a fragment of the R.equi genome containing the putative groEL2 gene.

A 4.713 kb fragment was found to contain a groEL2 gene (GenbankAccession No: AF233387) which was 1623 bp long and encoded a proteinwith a deduced molecular weight of 56543.5 Da. The gene had a high G+Ccontent of 68% which is not surprising as R. equi is known to possess aGC rich genome (Goodfellow, 1987).

Western immunoblot analysis indicated that R. equi heat shocked at 42°C. produced a protein approximately 60 kDa in size, which was notdetected in the culture grown at 30° C.

Homology of R. equi GroEL2 to Similiar Proteins in the Database

The inferred R. equi GroEL2 protein was found to be most closelyrelated, with approximately 90% identity, to the GroEL2 proteins ofMycobacterium tuberculosis.

Mycobacterium leprae, Mycobacterium avium and Tsukamurellatyrosinosolvens. It was also related to the GroEL2-like proteins fromother Gram positive actinomycetes such as Streptomyces albus,Streptomyces lividans and Streptomyces coelicor (Table 2). The R. equiGroEL2 was found to be less homologous (identity of approximately60-69%) to GroEL1 sequences of other actinomycetes and was approximately60% identical to GroEL sequences of organisms such as E. coli andHelicobacter pylori. TABLE 2 Similarity and identity of actinomyceteGroEL2 amino acid sequences to R. equi GroEL2 NCBI Accession Similarityto number of R. equi GroEL Identity to R. equi Organism GroEL2 proteinsequence GroEL sequence Tsukamurella AAB499990 93.2% 89.4%tyrosinosolvens Streptomyces 033658   89% 83.3% lividans StreptomycesCAB93056 90.3% 85.2% coelicor Streptomyces albus Q00798 90.5% 85.4%Mycobacterium PO9239 93.4% 89.6% leprae Mycobacterium PO6806 93.5%   90%tuberculosis H37RvDiscussion

R. equi-related organisms such as Mycobacterium and Streptomyces sp.contain two groEL genes (Rinke de Wit, et al., 1992). Of these groEL1 isconsidered to be part of the groE operon whereas groEL2 is usually foundat a different location on the chromosome (Duchêne. et al., 1994). TheR. equi gene sequenced was identified as a groEL2 gene for the followingreasons. Firstly, it was found to be homologous (90% identity) to otheractinomycete groEL2 genes. Further, it did not appear to have agroES-like gene upstream from it suggesting it was not part of a groEoperon. Previous studies on other R. equi related bacterial species suchas Mycobacteria and Streptomyces have shown a similar arrangement ofgroEL genes (Duchêne, et al., 1994, Rinke de Wit, et al., 1992). It islikely that R. equi contains at least two GroEL encoding genes one ofwhich is the monocistronic groEL2 sequenced in this study.

The high degree of identity that exists between the putative R. equiGroEL2 protein and the sequences of other actinomycete GroEL2 proteinsis hardly surprising, as heat shock proteins are known to be highlyconserved. Due to the highly conserved nature of these proteins and thegenes encoding them, they are often used in bacterial phylogeneticstudies (Gupta, 2000).

Example 2

Development of Vaccine Candidates Against R. equi

Construction of groEL2 Based DNA Vaccine

The groEL2 gene was PCR amplified and cloned into vector pcDNA3(Invitrogen) (Boshart, et al., 1985). The forward oligonucleotide primercontaining a start codon was 5′-ACGGTACCATGGCCAAGATCATCGC-3′ [SEQ ID No7] (KpnI site underlined; start codon in bold), the reverseoligonucleotide primer 5′-CTTCTAGACGGCGGATGCGAAATGC-3′ [SEQ ID No 8](XbaI site underlined). The forward primer also contained a Kozaksequence, CCATGG (start codon underlined) (Kozak, 1982).

PCR was performed using standard conditions at an annealing temperatureof 65° C., using a CsCl gradient purified preparation of pIMVS-Re1 astemplate and DyNAzyme™ EXT DNA polymerase (Finnzymes, Finland). The PCRproduct and pcDNA3 vector were digested separately with KpnI/XbaI andligated together. The construct designated pcDNA3-Re1 (FIG. 3) wascloned into E. coli DH5 cc α for vaccine preparation.

Another groEL2 based DNA vaccine candidate, one that did not contain anideal Kozak sequence was also constructed. This construct was created bydigesting pIMVS-Re1 with KpnI/XbaI in order to obtain a fragment(approximately 2 kb, from 2714 bp to 4710 bp) containing the groEL2gene. The fragment was ligated into KpnI/XbaI digested pcDNA3 vector.This construct was designated pcDNA3-hsp1 and was then cloned into E.coli DH5α for vaccine preparation.

Construction of vapA-Based DNA Vaccine

The vapA gene was PCR amplified and cloned into vector pcDNA3(Invitrogen) (Boshart, et al., 1985). The forward oligonucleotide primerwas: 5′-GA{overscore (GGATCC)}ATGGAGACTCTTCACAAGACG-3′ [SEQ ID No 9](BamHI site underlined; start codon in bold) the reverse oligonucleotideprimer was 5′-GAT{overscore (GAATTC)}TAACAACCGAGGCTGAGCG-3′ [SEQ ID No10] (EcoRI site underlined).

The forward primer also contained a Kozak sequence CC{overscore (ATG)}G(start codon underlined) (Kozak, 1982). PCR was performed using standardconditions at an annealing temperature of 65° C. using DyNAzyme™ EXT DNApolymerase (Finnzymes, Finland) and small scale plasmid extraction of R.equi ATCC 33701 as template. The PCR product and pcDNA3 vector wereseparately digested with BamHI/EcoRI and ligated together. The constructdesignated pcDNA3-Re2 was cloned into E. coli DH5 α for vaccinepreparation (FIG. 4).

Another vapA based DNA vaccine candidate not containing a modified Kozaksequence was also constructed. This construct was created by inserting aPCR amplified vapA gene containing restriction sites EcoRI and BamHIinto pcDNA3. The following oligonucleotides were used for PCRamplification of vapA: 5′-TCTTC{overscore (GGATCC)}GCTAATTACCGGC-3′ [SEQID No 11] (forward primer; BamHI site underlined) and 5′-G{overscore(GAATTC)}GCACCAATCCTGTTGCG-3′ [SEQ ID No 12] (reverse primer; EcoRI siteunderlined). Template used for the PCR reaction was a plasmid extractionof R. equi ATCC 33701 and PCR was performed using standard conditions atan annealing temperature of 55° C. using DyNAzyme™ EXT DNA polymerase(Finnzymes, Finland). Both the PCR product and pcDNA3 vector weredigested separately with EcoRI and BamHI and ligated together. Thisconstruct was designated pIMVS-vap1 and cloned into E. coli DH5 cc forvaccine preparation.

Strategy to Enhance the Immunogenicity of VapA

In order to increase the immunogenicity of VapA as vaccine, the VapAB-cell epitope encoding genetic sequence was inserted into groEL2 tocreate a chimeric groEL21vapA construct. This approach has been used byother researchers who have shown that in chimeric gene constructs, thecarrier gene acting as an adjuvant markedly enhances the immune responseto the inserted epitope, thus circumventing the need for conventionaladjuvants (Fomsgaard, et al., 1998).

The groEL2 gene was used as the carrier since previous studies haveshown that heat shock proteins as carriers in conjugated vaccines cansubstantially enhance a T-cell mediated immune response to theconjugated antigen (Barrios, et al., 1992).

The eukaryotic expression vector pcDNA3 was employed in the constructionof the DNA vaccines as it has been successfully used in other vaccinestudies (Todoroki, et al., 2000, Turnes, et al., 1999). Importantly, hisvector is known to be rich in immunostimulatory unmethylatedcytosine-phosphate-guanine dinucleotide (CpG) sequences thought topromote the efficacy of plasmid vaccines (Cohen, et al., 1998) (Sato, etal., 1996, Strugnell, et al., 1997). Furthermore, plasmid DNAadministered as an intramuscular vaccination is considered to activateCD4+ T-cells associated with a Th1 response (Leclerc, et al., 1997).

The protein vaccines used were tagged with histidine residues to enableconvenient purification (using Ni-NTA agarose) of the protein in itsnative form following its expression in E. coli and is an approach thathas been used by other researchers in the past for the preparation ofprotein vaccines (von Specht, et al., 2000).

Construction of Chimeric GROEL2/VAPA Based DNA Vaccine

The chimeric groEL2/vapA vaccine construct was prepared by the insertionof the immunogenic epitope NLQKDEPNGRA [SEQ ID No 3] of VapA into ahydrophilic region (as indicated by the Hopp and Woods hydrophobicityplot) and a predicted immunogenic epitope of GroEL (based upon studiescarried out on Salmonella typhi GroEL by Panchanathan) (Panchanathan, etal., 1998). The DNA sequence encoding the VapA epitope NLQKDEPNGRA [SEQID No 3], was inserted into groEL2 using overlap extension PCRmutagenesis (Ho, et al., 1989) (FIG. 6 and FIG. 7). Construct pcDNA3-Re1was used as a template in the initial (two) PCR reactions.

Oligonucleotide primers used in one of these reactions were5′-AACCTTCAGAAAGACGAACCGAACGGTCGAGCAGAGCGTCAGGAAGCGGTCC TCG-3′ [SEQ IDNo 13] oligonucleotide primer GVIF (sequence corresponding to VapAepitope to be inserted is underlined) and5′-CTATAGAATAGGGCCCTCTAGACGG-3′ [SEQ ID No 14]-oligonucleotide primerGVOR.

The other PCR was performed using oligonucleotide primers GVIR withsequence 5′-TGCTCGACCGTTCGGTTCGTCTTTCTGAAGGTTGGCGTCGGTCGCGAAGTACAGC G-3′[SEQ ID No 15] (sequence corresponding to VapA epitope to be inserted isunderlined) and oligonucleotide primer GVOF with sequence5′-GAGACCCAAGCTTGGTACCATGG-3′ [SEQ ID No 16] (Kozak sequenceunderlined).

The PCR products obtained from both the above reactions were separatedon a 1.5% agarose gel and purified using the QlAquick gel purificationKit (Qiagen, GmbH, Germany). Approximately 100 ng of each of the PCRproducts were used as the template in the final PCR reactions which wereperformed using oligonucleotide primers GVOF and GVOR (sequencesdescribed above).

All PCR reactions were performed using standard conditions at anannealing temperature of 59° C. The PCR product and pcDNA3 vector weredigested separately with KpnI/XbaI and ligated together. The constructwas designated pcDNA3-Re3 and was cloned into E. coli DH5α for vaccinepreparation (FIG. 8 and FIG. 9).

Preparation of DNA Vaccines

The vaccine constructs were propagated by growing a single colonycontaining the vaccine construct in a 10 ml aliquot of L-brothcontaining 100 μg/ml ampicillin for 6 hours at 37° C. with shaking. Thisculture was added to 500 nil L-broth containing 100 μg/ml ampicillin andgrown overnight at 37° C. with shaking. The following day a large-scaleplasmid extraction was performed. The plasmid extract was purified twiceby CsCl gradient centrifugation and then dialysed overnight twiceagainst 1×TE.

Prior to vaccine use, the plasmid preparation was processed usingstandard techniques (R. Strugnell, personal communication; protocol onDNA vaccine preparation, http://dnavaccine.com) as follows: NaCl (finalconcentration of 0.1M) and 2 volumes of absolute ethanol were added tothe plasmid solution and mixed. The preparation was then precipitated at−20° C. for 30 mins. The DNA was pelleted by centrifugation for 15 minsat 17, 000 g. The pellet was washed with 70% ethanol, air dried andresuspended in 1×PBS (volume of PBS used was half that of the originalvolume of DNA preparation treated).

The vaccine preparation was then treated as follows in order to removethe contaminating endotoxin (Manthorpe, et al., 1993): Triton X-114(TX-114) was added to the vaccine preparation to a final concentrationof 1% (v/v) and mixed. The mixture was left on ice for 5 mins, thenheated at 40° C. for 10 mins, allowing phase separation. The mixture wasthen centrifuged at 3000 g at 30° C. for 10 mins. The upper aqueousphase containing the DNA was removed and fresh TX-I 14 added and theextraction process was repeated twice. Finally, DNA was precipitatedwith the addition of an equal volume of isopropanol and centrifugationat 17,000 g, the DNA pellet was washed with 70% ethanol, dried andresuspended in 1×PBS (endotoxin free, Media Production Unit of theIMVS). The concentration of DNA was determined and was adjusted to 100μg/l by diluting in 1×PBS, thereafter, 100 μl aliquots of thepreparation were stored at −20° C. for vaccine use.

Endotoxin levels in the final vaccine preparations were confirmed to beless than 10 pg/ml by the QCL-1000 Limulus Amoebocyte Lysate Kit(BioWhittaker, Walkersville, Md., USA) (Li, et al., 1999) (test wasperformed by the Media Production Unit of the IMVS) prior to immunisingthe mice.

Expression of GROEL2-based DNA vaccine (pcDNA 3-Re1), VAPA-based DNAvaccine (pcDNA3-Re2) and chimeric GROEL2/VAPA DNA vaccine (pcDNA3-Re3)in Cos-7 cells Cos-7 cells were maintained in RPMI-1640 cell culturemedium containing L-glutamine (CSL, KS, USA) and 10% foetal bovine serum(Sigma Chemical Co.). Twenty-four hours prior to transfection, cellswere subcultured to ensure that they were in log growth phase.Approximately 3×10⁵ cells added to a Nunc™ 35 mm cell culture dish weretransiently transfected, with pcDNA3, pcDNA3-hsp1, pcDNA3-vap1,pcDNA3-Re1, pcDNA3-Re2 or pcDNA3-Re3 vectors using the following method:A 15 μl aliquot of Fugene™ transfection reagent (Boehringer Mannheim)was diluted in 85 μl serum free cell culture media and incubated for 5mins at room temperature. The preparations of each of the vaccineconstructs and pcDNA3 vector (5 μg of CsCl gradient purified plasmidpreparation) were added separately to the Fugene™ mixture and incubatedfor 15 mins at room temperature. The mixture was then added to the cellsin fresh media and incubated at 37° C. in an incubator, in the presenceof 5% CO₂ for 48 hours. Prior to harvesting, cells were checked forconfluence. The growth medium was transferred to a centrifuge tube, 1 mlof 1×PBS added to the cells prior to collecting them from the bottom ofthe cell culture dish. The cells were added to the same centrifuge tube.The tube was then centrifuged at 10,000 g to pellet the cells. Thepellet was washed by the addition of 1×PBS and centrifugation at 10,000g. The pellet was finally resuspended in a 50 μl aliquot of 1×PBS, mixedwith an equal volume of sample buffer and used in SDS-PAGE analysis andwestern immunoblot.

Preparation of a Plasmid Encoding His-Tagged VopA Protein

The vapA gene was amplified using PCR with a forward primer5′-GAGGATCCATGGAGACTCTTCACAAGACG-3′ [SEQ ID No 17] containing anlintroduced NcoI site (underlined) and reverse primer5′-GCCTCGAGGGCGTTGTGCCAGCTACC-3′ [SEQ ID No 18] containing an introducedXhoI site (underlined) for cloning into pET-28a (+) (Novagen) expressionvector. PCR was performed using standard conditions at an annealingtemperature of 65° C. with plasmid extraction of R. equi ATCC 33701 astemplate and DyNAzyme™ EXT DNA polymerase (Finnzymes, Finland). The PCRproduct and vector were separately digested with NcoI and XhoI andligated to create construct pIMVS-Re3 (FIG. 5).

His-tagged VapA Protein Preparation

His-tagged VapA from pIMVS-Re3 was essentially prepared using the methoddescribed for the production of His-tagged GroEL2, with the followingmodification: The protein was eluted from the Ni-NTA agarose using 100mM EDTA (EDTA was used as imidazole could not be used to successfullyelute the protein bound to Ni-NTA agarose). The protein eluate wasdialysed twice against 1×PBS but was not subjected to endotoxin removalusing TX-I 14 as this treatment was found to be ineffective for theremoval of endotoxin from the VapA protein preparation, possibly due tothe lipophilic nature of the protein. Endotoxin levels in the proteinpreparation were determined using the QCL-1000 Limulus Amoebocyte LysateKit (BioWhittaker, Walkersville, Md., USA) prior to use (testing carriedout by the Media production Unit of the IMVS) and varied between 100-500pg/ml. The His-tagged protein was separated on a 15% SDS PAGE gel anddetected in a western immunoblot using the VapA specific monoclonalantibody (Takai, et al., 1993a).

Construction of a plasmid expressing His-tagged GroEL2/VapA protein Themethod of preparation of His-tagged GroEL2/VapA protein expressingchimeric gene was essentially that used for the production ofgroEL2/vapA in pcDNA3-Re3, with the following modification: instead ofGVOR the following primer was used 5′-CGTCAAGCTTGAAGTCCATGCCGC-3′ [SEQID No 21] (HindIII site underlined), thereafter the His-taggedGroEL2VapA construct (pIMVS-Re4) was produced as described forpIMVS-Re2.

His-Tagged Chimeric GroEL21VapA Protein Preparation

The method of preparation of His-tagged GroEL2/VapA protein wasessentially as described for His-tagged GroEL2 production. The purifiedprotein was separated on a SDS PAGE gel and detected in a westernimmunoblot using GroEL2 specific monoclonal antibody.

Results

Expression of groEL2-Based DNA Vaccine in Cos-7 Cells

A large protein band of approximately 60 kDa was observed only in cellstransfected with pcDNA3-Re1 indicating the production of GroEL2. Thisprotein band was detected in western immunoblot analysis using theChlamydia trachomatis Hsp60 specific monoclonal antibody.

Expression of VAPA based DNA vaccine in Cos-7 cells

A protein band of approximately 19 kDa and a larger diffuse band ofapproximately 15 kDa were expressed in cells transfected with pcDNA3-Re2and was detected in western immunoblot analysis using VapA specificmonoclonal antibody (Takai, et al., 1993a). Protein expression was notobserved in Cos-7 cells transfected with pcDNA3-hsp2 (construct withoutan ideal Kozak sequence) or cells transfected with pcDNA3 vector. Theexpression of the 15 and 19 kDa proteins by pcDNA3-Re2 could not beobserved in Coomassie Brilliant Blue stained SDS PAGE but only in awestern immunoblot. This was not surprising as other workers havereported similar difficulty in visualising VapA on SDS PAGE usingCoomassie Brilliant Blue stain (S. Takai, personal communication).

Expression of GROEL2/VAPA-Based DNA Vaccine in Cos-7 Cells

A large protein band of approximately 60 kDa was expressed in cellstransfected with pcDNA3-Re3 (chimeric groEL2 IvapA vaccine construct).The protein was slightly larger than the GroEL2 protein expressed inCos-7 cells.

Example 3 The Immunogenicity of R. equi Specific Vaccines as Determinedin the Murine Model of Infection

Prior to vaccine use the protein preparations were processed as followsto make them suitable for in vivo use. All preparations were dialysedtwice against 1×PBS. Endotoxin was removed from the GroEL2 and chimericGroEL2/VapA protein preparations using TX-114. Endotoxin was not removedfrom the His-tagged VapA preparation as it was not possible tosuccessfully remove the endotoxin from this preparation using the TX-114based method.

Endotoxin levels were determined to be 100 pg/ml or less in theendotoxin treated protein preparations and around 100-500 pg/ml in theuntreated His-tagged VapA protein preparations using the QCL-1000Limulus Amoebocyte Lysate Kit (BioWhittaker, MD, USA).

Protein concentrations of the samples were determined using the Bioradprotein assay and samples were stored in 100 μl aliquots at −20° C.until required. Prior to vaccination the sample was thawed at roomtemperature and diluted to a concentration of 2 mg/ml in 1×PBS.

Preparation of R. equi for use in a Live Vaccine and Challenge Studies

R. equi strain ATCC 33701 was prepared for infection of mice usingpreviously described methods (Takai, et al., 1995a, Takai, et al., 1991a). Prior to use in the animal studies, the R. equi strain was confirmedfor the presence of the vapA gene by PCR and expression of VapA bywestern immunoblot. The strain was grown from an aliquot stored at −70°C. for 48 h in BHI broth, at 37° C. with shaking. Bacteria were pelletedby centrifugation at 10,000 g for 10 min washed once in 1×PBS anddiluted in sterile saline to obtain a suspension giving an OD ofapproximately 0.6 at 550 nm. This suspension was diluted by 50% insterile saline to obtain the final inoculum containing approximately1.5×10⁷ organisms in a 100 μl aliquot. The suspension was furtherdiluted in sterile saline to obtain a concentration of approximately 10⁵organisms for use as a live vaccine. The approximate numbers of bacteriawere confirmed in retrospect by plating an aliquot of the inoculum ontoHBA just prior to inoculating the mice and counting the coloniesfollowing 48 h incubation at 37° C.

Mice used in the Study

Groups of 6-8 week old female BALB/c mice were used (five in eachgroup). Animals were obtained from the Veterinary Services Division ofthe IMVS, (Gilles Plains, Adelaide, South Australia) and were certifiedto be specified pathogen free (SPF). Each group of mice was placed inseparate filter top cages following immunisation.

DNA vaccination of Mice

Each group of mice was vaccinated with pcDNA3-Re1, pcDNA3-Re2,pcDNA3-Re3 or the pcDNA3 vector (control group). An aliquot of 50 μg ofDNA (50 μl volume) was injected into each quadriceps muscle.

The animals were lightly anaesthetised via inhalation of Fluothane™(Halothane) (Zeneca, Cheshire, UK) before being vaccinated. This wasdone for easy injecting of the animals as well as to prevent theinjected DNA from being expelled by leg movement (muscle contraction).Animals were vaccinated on 3 occasions, 2 weeks apart.

Protein Vaccination of Mice

A 50 μl aliquot of the protein preparation containing a concentration of100 μg protein, was mixed with an equal volume of 1.3% aluminiumhydroxide gel (Alhydrogel, Asia Pacific Specialty Chemicals Ltd, NSW,Australia) to make up the 100 μl aliquot administered to each animal.Each group of mice was vaccinated with His-tagged GroEL2, chimericGroEL2/VapA or VapA protein preparations. A control group of mice werevaccinated with 100 μl of 1×PBS. Animals were vaccinatedintraperitoneally on 3 occasions, 2 weeks apart and bled prior to everyboost and just before challenge.

Live R. equi Vaccination

Groups of mice were immunised with sub-lethal doses of live R. equi forcomparison with the other vaccines. Animals were vaccinated withapproximately 10⁵ live R. equi strain ATCC 33701 administered by theintraperitoneal route. Animals were vaccinated on three occasions, twoweeks apart and bled prior to every boost and challenge. The number oforganisms used in the vaccine was chosen based upon previous studies(Takai, et al., 1999a). Prior to vaccination an aliquot of thepreparation was plated on HBA for retrospective determination of viablebacterial numbers in the preparation.

Co-administration of murine IL-12 encoding plasmid with DNA vaccinescandidates The murine cytokine IL-12 expressing plasmid pORF-mIL12(InvivoGen, CA, USA) (the plasmid is reported to secrete murine IL-12 bythe manufacturer and was used without any modification) waselectroporated into E. coli DH5a and DNA vaccine was prepared aspreviously described (see section on DNA vaccine preparation above) forintramuscular injection. The IL-12 insert contained the two murine IL-12subunits (p35 and p40) encoding genes linked by 2 bovine elastin motifs(10 amino acids long), creating a single IL-12 open reading frame,ensuring the same level of expression of both subunits (Lee, et al.,1998). An aliquot of 5 μg of this preparation was co-injected(intramuscular) along with the antigen as previously described.

Obtaining Serum Samples from Mice

Two weeks after every immunisation and just before challenge bloodsamples were obtained from the mice by retro-orbital eye bleed. Prior tobeing bled, the animals were lightly anaesthetised via inhalation ofFluothane™ (Halothane) (Zeneca, Cheshire, UK). Blood samples from eachgroup of mice were pooled (tubes containing the blood were incubated for30 min at room temperature and then for 1 h at −4° C., finally they werecentrifuged at 1000 g and the sera removed and stored at −20° C. untilrequired.

ELISA for the detection of total IgG and immunoglobulin subclasses IGG1,IGG2a, IGG2b The pattern of IgG subclass produced during an immuneresponse is widely accepted to be a reliable indicator of the type ofcytokines produced in that response. Generally, IgG2a is considered toreflect the IFN-γ response (associated with a cell-mediated response)while IgG1 isotype switching is promoted by IL-4 (cytokine associatedwith humoral immunity) (Mosmann and Coffman, 1989).

The determination of the levels of IgG and IgG subclasses were performedas follows: Nunc™ maxisorp plates were coated with 5 μg/ml (100 μlaliquot per well) His-tagged GroEL2 or VapA in coating buffer (Na₂CO₃ 15mM, NaHCO₃ 35 mM; pH 9.6) and used in an ELISA assay. Mouse serum wasdiluted 1 in 250 in PBS/0.05% Tween20 buffer containing 0.25 mg/ml E.coli extract (Promega, Wis., USA) and allowed to stand at roomtemperature for 30 mins prior to dispensing into the wells. The E. coliextract was used to aid in reduction of the background caused by thepotential cross-reaction of any E. coli-specific antibodies present inthe serum sample with the ELISA antigen. Secondary antibodies used wererabbit anti mouse IgG (H and L chain specific), γ2a, γ2b or yl chainspecific peroxidase conjugated affinity purified monoclonal antibodies(Rockland, Pa., USA) at working dilutions of 1 in 5000, 1 in 4000, 1 in5000 and 1 in 1000 respectively. The ODs of the reactions were read inan ELISA plate reader at a wavelength of 450 nm (reference wavelength630 nm).

The results were confirmed by western immunoblot using His-tagged VapAand GroEL2 proteins (results not shown).

Preparation of Antigen for DTH Response Studies

R. equi strain ATCC 33701 was grown for 48 h in 500 ml BHI broth at 37°C. with shaking. The culture was pelleted by centrifugation at 10,000 gfor 10 mins and washed twice in 1× PBS. The pellet was resuspended in200-500 μl 1×PBS. The suspension was sonicated on ice for 30 secs andboiled for 10 mins. The protein concentration was determined andadjusted to 100 μg/ml by diluting in 1×PBS. This preparation was storedat −20° C. until required.

DTH Response Studies

Two separate studies (on groups of three mice immunised with thedifferent vaccine candidates as described above) were carried out tomeasure DTH response in the hind footpads. The right hind footpad ofeach mouse was injected with 20 μl of the antigen and the correspondingleft hind footpad was injected with 20 μl 1× PBS. Footpad thickness wasmeasured at 24, 48 and 72 h intervals using Vernier calipers (theaverage of three readings ere obtained), DTH at 24 h was used in allanalyses as the reaction was most significant at his time compared withthe control. The percentage swelling was calculated using the followingformula:R. equi antigen footpad swelling (mm)-PBS footpad swelling/PBS footpadswelling×100Statistical Analysis of Data

Data were analysed using a Wilcoxon (rank sum) two sample test at asignificance level of P≦0.05. A non-parametric test was used as datawere found not to be normally distributed. The data were analysed usingSAS version 8.01 (SAS Institute, Inc. NC, USA).

Results

Symptoms Observed in Mice Following Challenge with R. equi

All animals except those immunised with the live R. equi vaccinedeveloped symptoms of mild illness 24 hours after challenge. Nosignificant weight loss was observed in these mice and none of theanimals succumbed to the infection. The mice appeared to be completelynormal by the fourth or fifth day after challenge.

Immune Response to Vaccination with Live R. equi

Mice vaccinated with live R. equi showed a Th1 biased immune response asindicated by moderately high IgG2a levels and low IgG1 levels. The IgG1,IgG2a and IgG2b responses increased with every boost. Interestingly, theVapA specific antibody response was higher than the GroEL2 specificresponse (Table 6.1). Importantly, a significant DTH response was alsodetected in these mice. Furthermore, the vaccinated mice showed enhancedclearance of R. equi following intravenous challenge.

Immune Response to GroEL2 Based Vaccine Candidates

Significant levels of IgG2a antibodies to the R. equi GroEL2 proteinwere detected in mice vaccinated with both the His-tagged GroEL2 proteinand DNA vaccine (pcDNA3-Re1), however, following two boosts the DNAvaccine was found to elicit a higher IgG2a response than the proteinvaccine (FIG. 10B). Both the IgG1 (FIG. 10A) and IgG2a antibody (FIG.

FIG. 10B) responses progressively increased with every boost. Followingthe last boost the IgG2b response (FIG. 1 IA) was lower than the IgG2aresponse and higher than the IgG1 response for the DNA based vaccine.

The addition of pORF-mIL12 increased the IgG1 response after the lastboost. In addition, following the last boost the IgG2b response was alsoincreased. The IgG2a response was less following the last boost. The DTHresponse in the pORF-mIL12 co-immunised group was lower than thatobtained with pcDNA3-Re1, indicating a lower Th1 bias than obtained withpcDNA3-Re1 alone.

The DTH responses induced by the DNA and His-tagged protein vaccineswere significant compared to the response in the mice immunised with thevector pcDNA3 (FIG. 11B). The response induced by the DNA vaccinepcDNA3-Re1 was higher than the response in the mice vaccinated withHis-tagged GroEL2.

Immune Response to VapA Based Vaccine Candidates

Moderate levels of IgG2a antibodies to the His-tagged VapA protein weredetected in mice vaccinated with vapA based DNA vaccine (pcDNA3-Re2).The level of IgG1 antibodies was much lower than the IgG2a levels (FIG.12A and FIG. 12B), indicating a Th1 biased immune response. The immuneresponse to the His-tagged VapA vaccine was a higher IgG2a and a muchhigher IgG1 response than with the DNA vaccine, possibly indicating aweaker Th1 type bias in immune response than that observed with the DNAvaccine. The IgG2b response (FIG. 13A) was lower than the IgG2a and wassimilar or higher than the IgG1 response with both the DNA and proteinvaccines tested, once again indicating a Th1 bias of the immuneresponse. These results indicate a Th1 bias in the immune responseelicited by the VapA based vaccines. The co-administration of pORF-mlLI2substantially increased the IgG1 responses to pcDNA3-Re2 and alsoincreased the IgG2a response but only until the last boost. Theco-administration of pORF-mIL 12 did not significantly alter the IgG2bresponse.

The DTH response of the mice vaccinated with vapA DNA vaccine(pcDNA3-Re2) and His-tagged VapA vaccines was significantly higher thanin the control mice (vaccinated with pcDNA3 alone) (FIG. 13B).

Immune Response to Chimeric GroEL2/VapA Vaccine Candidates

Significant levels of IgG2a antibodies to the R. equi GroEL2 proteinwere detected in mice vaccinated with the pcDNA3-Re3 (groEL2/vapAchimeric DNA vaccine) and chimeric GroEL/2NapA protein vaccines. Boththe IgG1 and IgG2a antibody response progressively increased with everyboost. The level of IgG1 antibodies was much lower than the IgG2a levels(FIG. 14A, FIG. 14B), indicating a Th1 bias in the immune response. TheIgG2b response (FIG. 14A) was lower than the IgG2a and higher than theIgG1 response both for the DNA based and protein vaccines tested,suggesting a Th1 bias to the immune response.

The DTH response of the mice vaccinated with pcDNA3-Re3 and His-taggedGroEL2/VapA was significantly greater than the control group micevaccinated with pcDNA3 vector (FIG. 15B).

The addition of pORF-mIL 2 increased the IgG1 response but the IgG2aresponse was not significantly altered following the last boost. TheIgG2b response was increased significantly following the final boost.

Assay to Detect Antibodies to the VapA B-Cell Epitope in the ChimericgroEL2/vapA Vaccine Construct.

Sera from mice immunised with the chimeric groEL2/vapA DNA vaccine(pcDNA3-Re3) were assayed to detect antibodies to the VapA B-cellepitope NLQKDEPNGRA [SEQ ID No 3]. This was carried using an ELISA withthe biotinylated peptide NLQKDEPNGRA [SEQ ID No 3] as the targetantigen. In addition, the His-tagged VapA was used as the target antigenin a separate ELISA. The OD values obtained with the sera from miceimmunised with the chimeric groEL2/vapA based vaccines were notsignificantly different from that obtained with the sera from thecontrol mice (results not shown). This suggests that the VapA epitopeinserted into GroEL2 did not elicit a detectable IgG response in themice.

Comparison of DNA Vaccines

Both the groEL2 (pcDNA3-Re 1) and chimeric groEL2/vapA (pcDNA3-Re3)vaccines produced significant total IgG, and specifically high IgG2a andDTH responses. The IgG1 and IgG2b responses were low to moderate.Generally, both the IgG1 and IgG2a responses were increased in thepresence of pORF-mIL 2. The responses generated by pcDNA3-Rc1 andpcDNA3-Re3 were generally similar, except for the IgG1 response whichwas higher with pcDNA3-Re1. Not surprisingly, both the IgG1 and IgG2aantibody response progressively increased with every boost.

The immune response generated by the vapA based DNA vaccines wassignificantly lower than the other DNA vaccines, when pORF-mIL12 wasco-injected there was a significant increase in IgG2a response followingthe first boost, however the response was still not as high as thatobserved with the groEL2/vapA (pcDNA3-Re3) chimeric vaccines. The IgG2a,IgG2b and IFN-γ response elicited by the groEL2 based vaccines(pcDNA3-Re1 and pcDNA3-Re3) were significantly higher than that observedwith the live vaccine suggesting a significantly higher Th1 type immuneresponse with the DNA vaccines. Contrary to this, the DTH responseobtained with the live vaccine was significantly higher than thatobserved with any of the plasmid vaccines (Table 3). Importantly, noneof the DNA vaccines elicited enhanced clearance in the mice unlike thelive vaccine.

Comparison of Protein Vaccines

Generally, all three His-tagged protein vaccines elicited apredominantly IgG2a response however, the IgG1 response produced wasalso substantially higher than the response observed with thecorresponding DNA vaccine candidates. This indicated a lower Th1 bias inthe immune response elicited by the protein vaccines when compared withthe DNA vaccines.

The His-tagged VapA vaccine elicited the strongest DTH response comparedwith the other protein vaccines tested. However, this result may bepartly caused by the presence of relatively high levels of endotoxin (asa consequence of being expressed in a Gram negative bacterium) in theVapA preparation compared with the other vaccines. Interestingly, eventhe His-tagged GroEL2 and chimeric GroEL2 vaccines produced asignificant DTH response that was higher than the response observed inthe live R. equi immunised mice (Table 3).

The groEL2 based DNA vaccines (pcDNA3-Re1 and pcDNA3-Re3) appeared toelicit a strong Th1 type immune response as indicated by the IgGsubclassing. In this regard, other reports have shown similar findingswith groEL2 based DNA vaccines developed to other bacterial pathogens(Noll, et al., 1994). Importantly, these vaccines elicited an immuneresponse that appeared to be more strongly Th1 biased than the vapAbased vaccine (pcDNA3-Re2), suggesting that groEL2 was possibly a betterDNA vaccine candidate than vapA.

The insertion of the VapA B-cell epitope NLQKDEPNGRA [SEQ ID No 3] intogroEL2 appeared to have enhanced the Th1 response elicited by GroEL2, asobserved in lower IgG1 and higher IgG2a responses.

The His-tagged protein vaccines unlike the corresponding DNA vaccinesdid not elicit a significant Th1 type immune response as indicated by ahigh IgG1 and IgG2a levels. Other researchers have reported similarfindings with regard to the use of protein vaccines for intracellularpathogens, which require a Th1 response in the host for clearance ofinfection (Turner. et al., 2000).

The groEL2 based DNA vaccine was found to elicit an immune response thatwas more strongly Th1 biased than the vapA based DNA vaccine, anindication that groEL2 is more immunogenic than vapA, when administeredas a DNA vaccine. TABLE 3 Summary of immune response to vaccination withgroEl2 based, vapA based and live R. equi vaccines DTH ELISA OD (450 nm)(mean ± SD) response at (antibody levels just prior to Vaccine 24 hours(% challenge) preparation swelling ± SD)⁺ Total IgG IgG1 IgG2a groEL2DNA 30 ± 10 1.353 ± 0.10 0.337 ± 0.05 1.445 ± 0.05 (pcDNA3-Re1)His-tagged GroEL2 35 ± 7 1.581 ± 0.05 0.396 ± 0.08 1.100 ± 0.058 proteinChimeric 30 ± 9 1.399 ± 0.12 0.084 ± 0.009 1.421 ± 0.19 groEL2/vapA DNA(pcDNA3-Re3) His-tagged 35 ± 7 1.468 ± 0.13 0.596 ± 0.02 1.413 ± 0.04GroEL2/VapA protein vapA DNA 28 ± 15 0.716 ± 0.13 0.035 ± 0.02  0.33 ±0.007 (pcDNA3-Re2) His-tagged VapA 45 ± 11 1.479 ± 0.10 0.590 ± 0.021.153 ± 0.08 protein Live R. equi (10⁵ 36 ± 5.2 VapA 0.019 ± 0.001*0.206 ± 0.02* organisms) specific 0.438 ± 0.019* GroEL2 specific 0.148 ±0.018** 0.136 ± 0.002** 0.186 ± 0.02** Control (pcDNA3 16 ± 4 0.134 ±0.019 0.013 ± 0.002 0.038 ± 0.021 vector) Control (1 × PBS)‡ 14 ± 80.104 ± 0.01 0.015 ± 0.002 0.034 ± 0.001⁺Preliminary studies indicated that DTH responses measured at 24 hoursafter footpad injection showed the greatest difference among the vaccinepreparations*VapA antigen specific response**GroEL2 antigen specific response‡As there was no significant difference between the overall response inPBS and pcDNA3 control mice, the PBS control data were not included infurther statistical analyses

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1. A chimeric protein, said chimeric protein being a GroEL protein,modification or analogue thereof having a surface exposed exogenousamino acid sequence inserted therein, the exogenous amino acid sequenceconfigured to elicit an immune response specifically reactive to theantigenic determinant.
 2. The chimeric protein as in claim 1 wherein theexogenous amino acid sequence is inserted in a hydrophilic region of theGroEL protein.
 3. The chimeric protein as in claim 1 wherein theexogenous amino acid sequence is inserted into a location of the GroELprotein comprising a GroEL antigenic determinant.
 4. The chimericprotein as in claim 2 wherein the GroEL is derived from R. equii.
 5. Thechimeric protein as in claim 4 wherein the hydrophilic region isselected from the group of hydrophobic regions consisting of V26-S54,V73-T90, G109-A155, M191-L246, R270-I290, G342-A 197, and V 415-N468. 6.The chimeric protein as in claim 4 wherein the hydrophilic region isM191-L246.
 7. The chimeric protein as in claim 1 wherein the exogenousamino acid sequence has a length of in the range of 3 to 25 amino acids.8. The chimeric protein as in claim 1 wherein the exogenous amino acidsequence has a length of about 11 amino acids.
 9. The chimeric proteinas in claim 1 wherein the exogenous amino acid sequence includes animmunodominant antigenic determinant of a pathogenic bacterial species.10. The chimeric protein as in claim 9 wherein the GroEL protein isderived from the pathogenic bacterial species.
 11. The chimeric proteinas in claim 9 wherein the pathogenic species is Rhodococcus equii andthe immunodominant antigenic determinant is derived from the Vap Aprotein.
 12. The chimeric protein as in claim 11 wherein the antigenicdeterminant is present in SEQ ID No
 2. 13. The chimeric protein as inclaim 11 wherein the antigenic determinant is present in SEQ ID No 3.14. A nucleic acid molecule including a chimeric protein encodingsequence and a control element positioned for expression of saidchimeric protein, said chimeric protein being a GroEL protein,modification or analogue thereof having a surface exposed exogenousamino acid sequence inserted therein, the exogenous amino acid sequenceconfigured to elicit an immune response specifically reactive to theantigenic determinant.
 15. The nucleic acid molecule as in claim 14wherein the exogenous amino acid sequence is inserted in a hydrophilicregion of the GroEL protein.
 16. The nucleic acid molecule as in claim14 wherein the exogenous amino acid sequence is inserted into a locationof the GroEL protein comprising a GroEL antigenic determinant.
 17. Thenucleic acid molecule as in claim 15 wherein the GroEL protein isderived from R. equi.
 18. The nucleic acid molecule as in claim 14wherein the exogenous amino acid sequence has a length in the range of 3to 25 amino acids.
 19. The nucleic acid molecule as in claim 14 whereinexogenous amino acid sequence includes an immunodominant antigenicdeterminant of a pathogenic bacterial species.
 20. The nucleic acidmolecule as in claim 19 wherein the GroEL protein is derived from thepathogenic bacterial species.
 21. The nucleic acid molecule as in claim19 wherein the pathogenic species is Rhodococcus equii and theimmunodominant antigenic determinant is derived from the Vap A protein.22. The nucleic acid molecule as in claim 21 wherein the antigenicdeterminant is present in SEQ ID No
 2. 23. The nucleic acid molecule asin claim 14 wherein the chimeric protein also includes a non GroELsequence that assists in the purification of the protein.
 24. Thenucleic acid molecule as in claim 23 wherein a plurality of histidineresidues are added to the C terminus of the chimeric protein.
 25. Thenucleic acid molecule as in claim 14 including a promoter for expressionin a host cell to elicit the immune response.
 26. The nucleic acidmolecule as in claim 25 said DNA molecule vector encoding aco-stimulatory molecule, said co-stimulatory molecule capable ofstimulating the immune response of the host.
 27. A method of elicitingan immune response in a mammal against an antigenic determinant themethod including the step of administering to the mammal a chimericprotein, the chimeric protein being a GroEL protein, modification oranalogue thereof having a surface exposed exogenous amino acid sequenceinserted therein, said exogenous amino acid sequence configured toelicit an immune response specifically reactive to the antigenicdeterminant.
 28. The method of eliciting an immune response as in claim27 wherein the exogenous amino acid sequence is inserted in ahydrophilic region of the GroEL protein.
 29. The method of eliciting animmune response as in claim 27 wherein the exogenous amino acid sequenceis inserted into a location of the GroEL protein comprising a GroELantigenic determinant.
 30. The method of eliciting an immune response asin claim 27 wherein the exogenous amino acid sequence has a length inthe range of 3 to 25 amino acids.
 31. The method of eliciting an immuneresponse as in claim 27 wherein exogenous amino acid sequence includesan immunodominant antigenic determinant of a pathogenic bacterialspecies.
 32. The method of eliciting an immune response as in claim 31wherein the GroEL protein is derived from the pathogenic bacterialspecies.
 33. The method of eliciting an immune response as in claim 31wherein the pathogenic species is Rhodococcus equii and theimmunodominant antigenic determinant is derived from the Vap A protein.34. The method of eliciting an immune response as in claim 33 whereinthe antigenic determinant is included in SEQ ID No
 2. 35. The method ofeliciting an immune response as in claim 28 wherein the GroEL protein isderived from R. equii.
 36. The method of eliciting an immune response asin claim 27 wherein the immune response includes an antibody responsespecific to the antigenic determinant.
 37. The method of eliciting animmune response as in claim 36 wherein the antibody response isproportionately greater for IgG2a in comparison to IgG1.
 38. The methodof eliciting an immune response as in claim 27 wherein the chimericprotein is administered in purified form in a pharmaceuticallyacceptable carrier.
 39. The method of eliciting an immune response as inclaim 38 wherein an adjuvant is co-administered.
 40. The method ofeliciting an immune response as in claim 27 wherein a nucleic acidmolecule capable of expressing the chimeric protein is administered tothe mammal so that on insertion into a cell of the mammal expression inthe host the chimeric protein is expressed in the cell.
 41. The methodof eliciting an immune response as in claim 40 wherein the nucleic acidalso expresses an immunostimulatory molecule in the cell.
 42. The methodof eliciting an immune response as in claim 40 wherein the nucleic acidis administered by intramuscular injection.